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PDBsum entry 1sgi
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Contents |
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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Molecular dissection of na+ binding to thrombin.
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Authors
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A.O.Pineda,
C.J.Carrell,
L.A.Bush,
S.Prasad,
S.Caccia,
Z.W.Chen,
F.S.Mathews,
E.Di cera.
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Ref.
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J Biol Chem, 2004,
279,
31842-31853.
[DOI no: ]
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PubMed id
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Abstract
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Na(+) binding near the primary specificity pocket of thrombin promotes the
procoagulant, prothrombotic, and signaling functions of the enzyme. The effect
is mediated allosterically by a communication between the Na(+) site and regions
involved in substrate recognition. Using a panel of 78 Ala mutants of thrombin,
we have mapped the allosteric core of residues that are energetically linked to
Na(+) binding. These residues are Asp-189, Glu-217, Asp-222, and Tyr-225, all in
close proximity to the bound Na(+). Among these residues, Asp-189 shares with
Asp-221 the important function of transducing Na(+) binding into enhanced
catalytic activity. None of the residues of exosite I, exosite II, or the
60-loop plays a significant role in Na(+) binding and allosteric transduction.
X-ray crystal structures of the Na(+)-free (slow) and Na(+)-bound (fast) forms
of thrombin, free or bound to the active site inhibitor
H-d-Phe-Pro-Arg-chloromethyl-ketone, document the conformational changes induced
by Na(+) binding. The slow --> fast transition results in formation of the
Arg-187:Asp-222 ion pair, optimal orientation of Asp-189 and Ser-195 for
substrate binding, and a significant shift of the side chain of Glu-192 linked
to a rearrangement of the network of water molecules that connect the bound
Na(+) to Ser-195 in the active site. The changes in the water network and the
allosteric core explain the thermodynamic signatures linked to Na(+) binding and
the mechanism of thrombin activation by Na(+). The role of the water network
uncovered in this study establishes a new paradigm for the allosteric regulation
of thrombin and other Na(+)-activated enzymes involved in blood coagulation and
the immune response.
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Figure 7.
FIG. 7. Stereo view of the Na^+ binding environment in the
structures of F (free fast form, gold), S (free slow form, red),
FL (PPACK-bound fast form, blue), and SL (PPACK-bound slow form,
green). Shown are all atoms within 3 Å of the bound Na^+
in the F structure, in addition to the side chains of Asp-189
and Asp-221. Note the similarity of the Na^+ coordination shell
between F and FL; the bound Na^+ is coordinated octahedrally by
the backbone O atoms of Lys-224 and Arg-221a and by four buried
water molecules that H-bond to (clockwise) Asp-189, Asp-221,
Gly-223, and Tyr-184a. Only some of these water molecules are
replaced in the absence of Na^+ (S and SL). Note the
rearrangement of the side chain of Asp-189 in the S structure
and the significant shift in the backbone O atom of Arg-221a
that assumes a position incompatible with Na^+ coordination.
H-bonds are shown by broken lines and refer to the F structure.
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Figure 8.
FIG. 8. Stereo view of the electron density maps of the S
(A), F (B), SL (C), and FL (D) intermediates of thrombin in the
regions bearing the most significant structural transitions.
Residues are rendered in CPK. The bound Na^+ is rendered as a
cyan ball. Shown are the 221–224 loop region and the 187–195
domain. Note how Asp-222 and Arg-187 have joined densities in
the F form, indicative of ion pair interaction, but not in the S
form. Also notable are the reorientation of Asp-189 and Glu-192
in the S form, as well as the shift in the position of Ser-195.
Other changes observed in the slow  fast transition involve
the network of water molecules (red balls) embedding the Na^+
site, the S1 pocket, and the active site region. In the fast
form, this network is well organized and contains 11 water
molecules. In the slow form, the water molecules are reduced to
seven, and the long range connectivity of the network is lost
(see also Fig. 9). The 2F[o] - F[c] electron density maps are
contoured at 0.7  for S and F and at 1.0
for
SL and FL.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2004,
279,
31842-31853)
copyright 2004.
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